Total proteins were obtained from treated melanoma cells using RIPA buffer and then subjected to protein quantification using a BCA kit (Pierce). Equal protein samples (50 μg) were loaded into and isolated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. The nonspecific binding sites in the membranes were blocked by incubating with TBST solution containing 5% milk for 2 h. After washing with PBS three times, the membranes were incubated with primary antibodies against DUSP5 (1:1000, ab200708, Abcam), TGFBR1 (1:1000, ab235178, Abcam) and GAPDH (1:10000, sc420485, Santa Cruz) overnight at 4 °C. On the following day, membranes were washed with PBS again three times and then incubated with HRP-conjugated secondary antibodies (1:2000, 4412S, Santa Cruz) for 2 h. Protein bands were detected by an Odyssey infrared imaging system (LI-COR).
Ab200708
Manufactured by Abcam
Sourced in United States
Ab200708 is a recombinant monoclonal antibody. It is designed for use in immunoassays, including but not limited to ELISA, immunohistochemistry (IHC), and Western blotting. The antibody is produced in vitro using recombinant technology.
Automatically generated - may contain errors
Lab products found in correlation
Ab235178, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Diaminobenzidine dab, by Gene Tech (1 mentions)
Ab194982, by Abcam (1 mentions)
A1978, by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab38898, by Abcam (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Bca protein assay kit, by Fdbio (1 mentions)
Ab49849, by Abcam (1 mentions)
A0216, by Beyotime (1 mentions)
Ripa buffer, by Fdbio (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
A0208, by Beyotime (1 mentions)
Abs137975, by Absin (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Super signal west femto 34095, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ab210498, by Abcam (1 mentions)
8 protocols using ab200708
1
Western Blot Analysis of Melanoma Proteins
2
Immunohistochemical Analysis of DUSP5 in Thyroid Cancer
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The tissue microarrays (TMAs) were purchased from US Biomax, Inc. (TH8010a Rockville, MD, USA), which included 20 FTC and 44 PTC specimens. The clinical information of the specimens was provided by the manufacturer. Human TC tissues, obtained from Qilu Hospital of Shandong University, also were verified expression of DUSP5.
Tissue paraffin sections were cut at a thickness of 4 μm. They were then deparaffinized in xylene and rehydrated through graded alcohol, and heat induced antigen retrieval used Tris‐EDTA buffer (1 mM EDTA, 10 mM Tris, 0.05% Tween 20, pH 9.0) for 20 minutes. The slides were incubated with 3% H2O2 for 10 minutes before blocking with 5% goat serum, in order to block the activity of endogenous peroxidase. The slides were incubated with dual‐specificity phosphatase 5 (DUSP5) (1:200, ab200708, Abcam, Cambridge, USA) overnight at 4°C. After washing with PBS, the slides were incubated with peroxidase enzyme‐conjugated goat anti‐rabbit secondary antibody (#PV9001, 1:400, Zhongshan, Beijing, China) for 30 minutes at 37°C. Results of immunohistochemical staining were recorded as an average positive area (yellowish‐brown color area) by using Diaminobenzidine tetrahydrochloride (ZSBIO, Beijing, China). The slides were then counterstained with hematoxylin.
Tissue paraffin sections were cut at a thickness of 4 μm. They were then deparaffinized in xylene and rehydrated through graded alcohol, and heat induced antigen retrieval used Tris‐EDTA buffer (1 mM EDTA, 10 mM Tris, 0.05% Tween 20, pH 9.0) for 20 minutes. The slides were incubated with 3% H2O2 for 10 minutes before blocking with 5% goat serum, in order to block the activity of endogenous peroxidase. The slides were incubated with dual‐specificity phosphatase 5 (DUSP5) (1:200, ab200708, Abcam, Cambridge, USA) overnight at 4°C. After washing with PBS, the slides were incubated with peroxidase enzyme‐conjugated goat anti‐rabbit secondary antibody (#PV9001, 1:400, Zhongshan, Beijing, China) for 30 minutes at 37°C. Results of immunohistochemical staining were recorded as an average positive area (yellowish‐brown color area) by using Diaminobenzidine tetrahydrochloride (ZSBIO, Beijing, China). The slides were then counterstained with hematoxylin.
3
Immunohistochemical Analysis of DUSP5 and HPV16 E7
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Paraffin-embedded tissue slides (3 μm thick) were heated at 65°C for 30 min and deparaffinized in xylene. After rehydration in a graded series of ethanol solutions, slides were immersed in 0.01 M sodium citrate buffer (pH 6.0) inside a pressure-cooker for 15 min to achieve heat-induced antigen retrieval. Tissues were then incubated with 3% hydrogen peroxide for 30 min, rinsed with PBS, and incubated with 5% nonimmune goat serum for 30 min inside a moist chamber. Tissues were subsequently incubated overnight with anti-DUSP5 (ab200708, 1 : 500; Abcam) or anti-HPV16 E7 (1 : 500) at 4°C. Finally, the tissues were washed and incubated with secondary anti-rabbit IgG (Gene Tech, China) for 30 min, followed by staining with diaminobenzidine (DAB, Gene Tech) and hematoxylin. Images were obtained with a KFBIO Digital Slide Viewer.
4
Western Blot Protein Quantification
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Protein concentrations in cell extracts were determined by Bio‐Rad DC Protein Assay kit (Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Equal amounts of protein fractions of lysates were resolved over 10% SDS‐PAGE and transferred to PVDF membrane (EMD Millipore, Billerica, MA, USA). The membranes were incubated with rabbit anti‐DUSP5 (1:1000, ab200708, Abcam, Cambridge, USA), rabbit anti‐E cadherin (1:1000, ab194982, Abcam, Cambridge, USA, rabbit anti‐Vimentin (1:1000, ab92547, Abcam, Cambridge, USA), rabbit anti‐MMP9(1:500, ab38898, Abcam, Cambridge, USA), mouse anti‐β‐actin (1:1000; A1978, Sigma‐Aldrich; Merck KGaA) overnight at 4°C. The next day, corresponding peroxidase‐labeled goat anti‐rabbit and anti‐mouse secondary antibody (1:10 000) were used as the second antibody, respectively. The peroxidase activity was detected using the FluorChem E enhanced chemiluminescent system (ProteinSimple, San Jose, CA, USA). And ImageJ software (National Institutes of Health, USA) was used to quantify the densitometer of the optical density of the bands
5
Western Blot Analysis of AKAP12 and DUSP5
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Proteins were extracted from corresponding NHEKs using RIPA buffer (Fdbio science, Hangzhou, China) containing 1% PMSF for 15 min, followed by centrifugation for 10 min at 14,000 rpm at 4°C. The protein content was measured with a BCA Protein Assay kit (Fdbio science, Hangzhou, China). After denaturing at 100°C for 10 min, protein samples were separated on a SDS-polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF, Bio-Rad) membrane. After blocking with 5% skimmed milk in Tris-Buffered Saline containing 0.5% (v/v) Tween-20 (TBST), the membrane was incubated with primary antibody at 4°C overnight. The commercial antibodies used as primary antibody were anti-AKAP12 (ab49849, 1:1000; Abcam, Cambridge, MA, United States), anti-DUSP5 (ab200708,1:1000; Abcam, Cambridge, MA, United States) and anti-beta actin (abs137975,1:5000; Absin, Shanghai, China). After washing with TBST, the membrane was further incubated with HRP-labeled goat anti-mouse IgGs (A0216, 1:5000; Beyotime, Shanghai, China) or HRP-labeled goat anti-rabbit IgGs (A0208, 1:5000; Beyotime, Shanghai, China) at 37°C for 2 h. Beta actin was used as a loading control for Western blotting.
6
Western Blot Analysis of DUSP5 in Activated T Cells
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Spleen and lymph node tissues were isolated from Dusp5-/- and C57BL/6 WT mice as described above. CD8+ CD44- naïve T cells were purified from these tissues, then activated and differentiated. Cells were differentiated in high IL-2 SLEC media as DUSP5 is difficult to detect in unstimulated tissues and IL-2 is the strongest inducer of DUSP5 expression. Whole cell lysates were isolated in Radioimmunoprecipitation assay (RIPA) buffer and 25μg of protein was separated on a 4–20% gradient SDS-PAGE gel. After transfer to PVDF membranes, the blots were blocked in 5% bovine serum albumin (BSA) in Tween-20 tris-buffered saline (TBST) (0.1% Tween-20 in TBS) for 1 hour and incubated overnight at 4°C with monoclonal antibody against DUSP5 (1:1000; Abcam, AB200708) and for 1 hour at room temperature with monoclonal antibody against β-actin (1:10,000; Sigma, A5441). HRP-conjugated mouse-anti-sheep and anti-rabbit secondary antibodies were used at 1:10,000 dilutions. Blots were incubated with a 1:100 mixture of Femto:Pico substrates (SuperSignal West Femto, 34095, Thermo Scientific, SuperSignal West Pico, 34078, Thermo Scientific) and exposed using a ChemiDoc Touch imaging system (Bio-Rad, Hercules, CA).
7
Western Blot Analysis of Protein Markers
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We prepared protein lysates using RIPA lysis buffer. Protein concentration was tested with BCA Protein Quantitation Kit (Beyotime, Shanghai, China). Protein aliquots were separated through 10–12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Boston, MA, USA), which were blocked using 5% defatted milk (Bio-Rad, Hercules, CA, USA) under ambient temperature for 1 hour and incubated using primary antibodies overnight under 4°C: TFAP2C (AV38284, 1:1,000; Sigma), DUSP5 (ab200708; 1:1,000; Abcam), p62 (ab56416, 1:1,000; Abcam), LC3 (#4108, 1:1,000; Cell Signaling Technology), Beclin1 (ab210498, 1:1,000; Abcam), Bax (ab182733, 1:2,000; Abcam), Bcl-2 (MA5-11757, 1:1,000; Invitrogen), GAPDH (ab9485, 1:1,000; Abcam), followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody (#7074, 1:1,000; Cell Signaling Technology) for 1 hour at room temperature. Signals were visualized using SuperSignal Pico PLUS chemiluminescent substrate (Pierce, Rockford, IL, USA). GAPDH served as a loading control. Data analysis was conducted using ImageJ software (NIH, Bethesda, MD, USA).
8
HCEC Lysate Preparation and Western Blot
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HCEC lysates were prepared using RIPA buffer (r0010; Santa Cruz Biotechnology, Dallas, TX, USA) supplemented with 1% phenylmethylsulfonyl fluoride. A volume of 50 µL lysate was added into each well of a 24-well plate and incubated for 15 minutes on ice. The lysate was collected by scraping the cells with a 1 mL pipette tip and transferred to a 1.5 mL centrifuge tube. After centrifugation at 12,000 rpm for 20 minutes at 4°C, the supernatant was collected and mixed with 5 × loading buffer. The mixture was then heated at 95°C for 10 minutes. The sample was either directly used for Western blot or stored at −80°C for future use.
For Western blot analysis, protein samples were subjected to 10% acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for antibody incubation. Dusp5 antibody (1:1000, ab200708; Abcam) was used, and GAPDH antibody (1:1000, LK9002; Tianjin Sungene Biotech Co., Ltd, Tianjin, China) was used as a loading control. The protein bands were quantified by ImageJ grayscale image peak analysis, which involved selecting the bands with the rectangle tool after the images were inverted to the 8-bit format.
For Western blot analysis, protein samples were subjected to 10% acrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes for antibody incubation. Dusp5 antibody (1:1000, ab200708; Abcam) was used, and GAPDH antibody (1:1000, LK9002; Tianjin Sungene Biotech Co., Ltd, Tianjin, China) was used as a loading control. The protein bands were quantified by ImageJ grayscale image peak analysis, which involved selecting the bands with the rectangle tool after the images were inverted to the 8-bit format.
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